Transcript powerpoint

Bacterial vaginosis uncovered:
Aetiology, complications and
practical implications
Dr Janet Wilson
Leeds Teaching Hospitals NHS Trust, UK
Conflicts of Interest
Conflicts of Interest
I have received unconditional research grants in the
form of diagnostic tests from Hologic/GenProbe
I have received a speaker honorarium from BD
Diagnostics
I have participated in BV research studies sponsored
and funded by Starpharma
Prevalence of bacterial vaginosis
Commonest cause of abnormal discharge in women
of child bearing age
In UK: General practice
Antenatal clinic
Pre TOP clinic
Attending STI clinic
9%
15%
15-30%
25-35%
50% women are asymptomatic (complications still)
What is BV – aetiology 1950s
• Gardner and Dukes discovery of Haemophilis vaginalis in 1955
• They hypothesised that Gardnerella
vaginalis was the specific sexually
transmitted aetiological agent of BV
• Original experiments by Gardner and Dukes:
– 1/13 women inoculated with G. vaginalis developed BV
– 11/15 inoculated with vaginal material from infected
women developed BV
Gardner et al. Am J Obstet Gynecol 1955
Aetiology – later
th
20
century
20th Century polymicrobial theory from bacterial cultures
•Overgrowth of Gardnerella vaginalis, anaerobic bacteria,
Mycoplasma hominis, Ureaplasma urealyticum, and Mobiluncus
species, which replace the lactobacilli
•Bacteria increased 100-1000 times
•G. vaginalis present in many women with
normal flora and in virgins - not considered
main cause or to be sexually transmitted
•Treatments covering anaerobic bacteria such as metronidazole
and clindamycin work best
•Suggests more central role than Mobiluncus and mycoplasmas
•Symbiotic relationship between P. bivia and G. vaginalis
Exact trigger factor and role of bacteria unknown
Aetiology – 21st century
21st Century polymicrobial +++ theory
from molecular techniques
• Using bacterial 16S ribosomal DNA amplification
newly identified organisms associated with BV:
– Atopobium vaginae, Megasphaera,
Sneathia, Leptotrichia, Clostridiales order –
BVAB 1, 2, 3
Fredricks et al. NEJM 2005
• Detection of Megasphaera, A. vaginae, BVAB-1,
BVAB-3, highly sensitive and specific for BV
Fredricks et al. J Clin Microbiol 2007
But no new prime aetiological bacteria
identified – cause still unknown
Fluoresence in-situ
hybridisation (FISH)
Aetiology – 21st century
21st Century polymicrobial +++ theory
Using bacterial 16S ribosomal DNA amplification
different patterns in vaginal microbial diversity
identified
Geographical variation reflecting different
ancestries
In US women five vaginal microbiota groupings
(termed community state types, CST)
identified
Four dominated by different lactobacilli species,
fifth depleted of LB and resembled BV
CST associated with ethnicity – African-American
and Hispanic women more likely to be LB
depleted Ravel et al. Proc Natl Acad Sci USA 2011
Heatmap showing distribution
of bacteria in vaginal microbial
communities of 394 US women
Questions what are normal flora?
Aetiology still not explained and appearing more complex!
Aetiology –
st
21
century
Discovery of biofilms on vaginal biopsies
40 women without BV, 20 women with BV
• Loosely dispersed bacteria (Lactobacilli and
aerobes) in those without BV
• Dense biofilm of G. vaginalis in women with BV
• A. vaginae also present in 80% of biofilms
Only G. vaginalis developed a biofilm specific to BV
Swidsinski et al. Obstet Gynecol 2005
G. vaginalis is the adherent Gram-negative
bacteria on clue cells
Cook et al. J Infect Dis 1989
Aetiology of BV
G. vaginalis either:
Dispersed
Cohesive
= no BV
= BV and biofilm
Women with:
Dispersed Gardneralla: all normal vaginal flora
Cohesive Gardnerella: 10 BV on Gram stain, 2 intermediate
No Gardnerella: 2 BV, 2 intermediate, 48 normal flora
Cohesive Gardnerella had sensitivity 83%, specificity 97%
Swidsinski et al. Gynecol Obstet Invest 2010
G. vaginalis colonises 50-70% of women with normal vaginal
flora in small amounts
Srinivasan et al. PLoS One 2012
This is probably colonisation by dispersed G. vaginalis
Aetiology of BV
Are dispersed and cohesive G. vaginalis different strains?
G. vaginalis from BV versus G. vaginalis from normal flora:
• Better adherent to epithelial cells and cytotoxic (non-BV
was not) and more aggregative and produced a thick
biofilm
Harwich et al. BMC Genomics 2010
• Only BV-associated isolates able to adhere in high density
clusters to HeLa cells
Castro et al. Sci Rep 2015
• BV associated G. vaginalis produce mucinase
Yeoman et al. Plos One 2011
• Only two genotypes of G. vaginalis produce sialidase
Lopes Dos Santos Santiago et al. Am J Obstet Gynecol 2011
Conclusion: Differences in metabolic and virulence potential
Aetiology of BV
Are dispersed and cohesive G. vaginalis different strains?
G. vaginalis phenotypic heterogeneity known for years
Sequence-based analyses identified 4 distinct molecular
subgroups with high level of diversity - clades 1-4 - but
possibly different species?
Ahmed et al. J Bacteriol 2012
60 clinical samples from chronic vaginitis clinic:
94-97% were positive for G. vaginalis but only 37% had BV
BV associated with clades 1 and 3; intermediate with clade 2
No association with BV and clade 4
Balashov et al. J Med Micro 2014
Aetiology of BV
Are dispersed and cohesive G. vaginalis different strains?
They probably are – possibly even different species
Int J STD AIDS 2008
Back to single pathogen hypothesis of 1955
Could pathogenic cohesive biofilm-forming G. vaginalis
be responsible for BV?
Aetiology of BV
Unlike other anaerobes G. vaginalis possesses the three key
virulence determinants to cause BV:
• Adherence to vaginal epithelial cells
• Capacity to produce biofilms
• Cytotoxic activity
Patterson et al. Microbiology 2010
Alves et al. J Infect Dis 2014
Single species starts process of biofilm scaffolding in the
mouth to which other species adhere – probably same
mechanism in the vagina
Does cohesive G. vaginalis start the vaginal biofilm
to which the other BVAB attach?
Is this the sexually transmitted agent of BV?
New model of BV pathogenicity
Normal
vaginal flora
Lactobacilli
New model of BV pathogenicity
Sexual transmission
of cohesive (biofilm
forming) G. vaginalis
Normal
vaginal flora
Lactobacilli
New model of BV pathogenicity
Sexual transmission
of cohesive (biofilm
forming) G. vaginalis
Normal
vaginal flora
Lactobacilli
Adherence to
epithelial cells
Displacement
of lactobacilli
Adherence and displacement of LB
L. crispatus produces bacteriocins and core proteins - reduce
ability of G. vaginalis to adhere to epithelial cells
Ojala et al BMC Genomics 2014
G. vaginalis produces bacteriocins
active against LB
Teixeira et al. J Med Microbiol 2010
Cohesive G. vaginalis displaced L. crispatus from epithelial cells
Castro et al. Int J Med Sci 2013
G. vaginalis most pathogenic of BVAB as defined by highest
initial adhesion
Patterson et al. J Infect Dis 2014
G. vaginalis greatest capacity of BVAB to adhere in presence of
L. crispatus
Machado et al. Int J Mol Sci 2013
New model of BV pathogenicity
Sexual transmission
of cohesive (biofilm
forming) G. vaginalis
Adherence to
epithelial cells
Displacement
of lactobacilli
Normal
vaginal flora
Lactobacilli
Replication of
G. vaginalis
and other BVAB
Replication and biofilm formation
Symbiotic relationship between P. bivia and G. vaginalis
Pybus et al. J Infect Dis 1997
Growth benefit to G. vaginalis
from addition of any second
species
Machado et al. Int J Mol Sci 2013
G. vaginalis most pathogenic of BVAB as defined by greatest
propensity to form a biofilm
Patterson et al. J Infect Dis 2014
G. vaginalis biofilm can withstand 4-fold higher concentrations
of lactic acid and hydogen peroxide than dispersed G. vaginalis
Patterson et al. Am J Obstet Gynecol 2007
New model of BV pathogenicity
Sexual transmission
of cohesive (biofilm
forming) G. vaginalis
Adherence to
epithelial cells
Displacement
of lactobacilli
Normal
vaginal flora
Lactobacilli
Replication of
G. vaginalis
and other BVAB
Formation of biofilm
along with other BVAB
Biofilm protects against
lactic acid and H2O2
New model of BV pathogenicity
Sexual transmission
of cohesive (biofilm
forming) G. vaginalis
Adherence to
epithelial cells
Displacement
of lactobacilli
Normal
vaginal flora
Lactobacilli
Replication of
G. vaginalis
and other BVAB
Established BV
Formation of biofilm
along with other BVAB
Biofilm protects against
lactic acid and H2O2
New model of BV pathogenicity
Sexual transmission
of cohesive (biofilm
forming) G. vaginalis
Adherence to
epithelial cells
Displacement
of lactobacilli
Normal
vaginal flora
Lactobacilli
Replication of
G. vaginalis
and other BVAB
Established BV
Treatment
Formation of biofilm
along with other BVAB
Biofilm protects against
lactic acid and H2O2
New model of BV pathogenicity
Sexual transmission
of cohesive (biofilm
forming) G. vaginalis
Adherence to
epithelial cells
Displacement
of lactobacilli
Normal
vaginal flora
Lactobacilli
Replication of
G. vaginalis
and other BVAB
Established BV
Treatments have
high failure and
recurrence rates
Formation of biofilm
along with other BVAB
Biofilm protects against
lactic acid and H2O2
Complications of BV
High rate of recurrence -is it sexually transmitted?
STI and HIV acquisition/transmission
Association with PID
Pregnancy complications
Frequency of BV recurrence
121 women successfully treated with oral
metronidazole. Follow up 12 months
Recurrence rates:
23% 1 month
43% 3 months
58% 12 months
Bradshaw et al. J Inf Dis 2006
BV and sexual transmission?
Recurrence 2-3 fold higher if same regular sex partner after
treatment compared with change in partner
Bradshaw et al. J Inf Dis 2006
Male contacts of women who developed BV had significantly
more female partners in last 30 days than those without BV
Schwebke et al. Sex Transm Dis 2005
Consistent condom use strongly protective against BV
Ad OR 0.37 (95% CI 0.20-0.94) Hutchinson et al. Epidemiology 2007
No prevalent or incident BV in young women without coital or
non-coital sexual experience
Fethers et al. J infect Dis 2009
50 premenarche girls: 10% had dispersed G. vaginalis; none
had cohesive (biofilm associated)
Swidsinki et al. Gyneco Obstet Invest 2010
Evidence of transmission in WSW
Original experiments by Gardner and Dukes:
– BV occurred in 8% inoculated with G. vaginalis but in
73% inoculated with vaginal material from infected
women
In WSW significant concordance of vaginal flora in couples
43%
95%
87%
Berger 1995
Marrazzo 2002
Evans 2008
BV associated with partner with BV and sharing sex toys in
WSW ie practices that transmit vaginal fluid
Marrazzo et al. Sex Transm Dis 2010
Concordance of G. vaginalis in partners
Gardner and Dukes isolated G. vaginalis from urethra of 96%
of male partners of women with BV
Vaginal cultures from women with BV, urethral culture (within
24 hours) from male partners - G. vaginalis biotypes
identical in 92%
Piot et al, J Clin Microbiol 1984
Molecular typing of G. vaginalis showed male/female sexual
partners shared the same strains
Eren et al. PLoS One 2011
All male partners of women with cohesive G. vaginalis had
cohesive G. vaginalis
No concordance in male partners of women with dispersed G.
vaginalis
Swidsinski et al. Gynecol Obstet Invest 2010
Suggests cohesive G. vaginalis is the sexually transmitted
agent of BV
BV and acquisition of STIs
Increased acquisition with BV:
GC and CT
TV
HSV-2
HSV-2 shedding
1.5-2.0 fold
9-fold
2.1 fold
2.3 fold
Brotman et al. J Infect Dis 2010
Rathod et al. Sex Transm Dis 2011
Cherpes et al. Clin Inf Dis 2003
Cherpes et al. Clin Inf Dis 2005
Treatment then prophylaxis asymptomatic BV versus
observation
Prophylaxis
1.58 (95% CI 1.20-1.87) ppys
Observation
2.29 (95% CI 1.95-2.63) ppys
Schwebke et al. Am J Obstet Gynecol 2007
Treatment then RCT monthly metronidazole gel or
placebo
Almost 50% reduction of CT/NG/MG over 12 months
with monthly presumptive treatment
Balkus et al. JID 2016
BV and HIV acquisition
Meta-analysis of 23 studies; 30 739 women
HIV incidence with BV
RR 1.6 (95% CI 1.2-2.1)
- low HIV risk
RR 2.3 (95% CI 1.7-3.2)
- high HIV risk
RR 1.4 (95% CI 1.5-1.8)
BV prevalence 30% with RR 1.6; Population attributable risk 15%
Atashili et al. AIDS 2008
HIV positive women: Prevalence of BV OR 1.29 (1.08-1.55)
Persistence of BV OR 1.49 (1.18-1.89)
Immunocompromised women had more severe BV on Gram-stain
OR 1.50 (1.12-2.00)
Jamieson et al. Obstet Gynecol 2001
BV and HIV transmission
Heterosexual transmission
Prospective study of 2236 HIV-1+ve women and uninfected male
partners (part of large HIV/HSV trial)
50 incident infections (sequencing proven transmissions)
• HIV-1 incidence with BV
2.91/100 person years
• HIV-1 incidence normal flora
0.76/100 person years
• Adjusted Hazard ratio
3.17 (95% CI 1.37-7.33)
Cohen et al. PLoS Medicine 2012
Vertical transmission
16S ribosomal DNA amplification of vaginal secretions from 10
transmitters and 54 non-transmitters
MTCT of HIV associated with altered vaginal microbiota
Gardnerella vaginalis significantly associated with HIV MTC
transmission - Adjusted OR 1.7; P = 0.004
Frank et al. J Acquir Immune Defic Synr 2012
BV and pelvic inflammatory disease
Cross-sectional studies have reported significant association between PID and
BV but cause or effect?
Cervical and endometrial samples for BVAB from 545 women enrolled in PID
Evaluation and Clinical Health (PEACH) study (no metronidazole given)
A. vaginae, BVAB1, Sneathia sanguinegens, S. amnionii significantly associated
with BV
Endometritis at presentation:
All BVAB Ad OR 2.0 (1.0-4.0)
Endometritis 30 days after treatment:
All BVAB Ad OR 5.7 (1.4-23.3)
(Excluding women with CT and NG)
All BVAB Ad OR 8.5 (1.6-44.6)
Recurrent PID:
All BVAB Ad OR 3.9 (1.9-8.2)
Infertility:
All BVAB Ad OR 3.8 (1.4-10.2)
Antibiotic sensitivities of many of BVAB not known but supports including cover
against anaerobes in PID treatment
Haggerty et al. Sex Transm Infect 2016
BV and pregnancy
Cohort studies Meta-analysis of 18 studies; 20 232 women
Preterm birth
Overall risk
2.19 (1.54-3.12)
BV <16 weeks
7.55 (1.80-31.65)
BV <20 weeks
4.20 (2.11-8.39)
Spontaneous abortion
Overall risk
9.91 (1.99-49.34)
Postnatal infection
Overall risk
2.53 (1.26-5.08)
Leitich et al. Am J Obstet Gynecol 2003
BV common infection; population attributable risk of PTB due
to BV estimated to be 30% in USA at cost $1 billion
Koumans et al. Sex Transm Dis 2001
Practical Implications
Treatment of BV 20th & 21st Centuries
No new additions to treatment guidelines for years
Metronidazole
Clindamycin
Clindamycin
Tinidazole
Oral 500mg bd for 7 days
Vaginal gel 5gm for 5 days
Vaginal cream 5gm for 7 days
Oral 300mg bd for 7 days
Oral 2g daily for 2 days
Oral 1g daily for 5 days
Cure rates at one
month 71-88%
Clindamycin and metronidazole same efficacy irrespective of
regimen
Oduyebo et al. Cochrane Reviews 2009
14 days metronidazole same relapse rate as 7 days
Schwebke et al. Clin Inf Dis 2007
Clindamycin + metronidazole cure/recurrence rates same as
metronidazole alone
Bradshaw et al. PLoS One 2012
Bacterial Response to Treatment
With successful treatment 3-4 log reduction of Gardnerella
and BVAB. No significant change in those not cured
Fredricks et al. J Clin Microbiol 2009
Treatment RCT of clindamycin versus metronidazole
Treatment failures ≤1 month retreated with same treatment clinical cure rate equal to primary responder
Suggests antimicrobial resistance is not the problem
Bunge et al. Sex Transm Dis 2009
Treatment trials of male partners
5/6 RCTs of treatment of male sexual partner - no improvement in cure rate,
or reduction in recurrence rate
1/6 short term (<8 weeks) improvement with metronidazole
Potter J. Br J Gen Pract 1999
All 6 had significant flaws
5/6 published before 1996 CONSORT statement, other in 1997
Eligibility criteria for ‘partners’ not given or duration of sex
Power calculations inadequate; Insufficient randomisation methods
Treatment adherence in females and males not reported
5/6 treatment suboptimal metronidazole 2gm stat - 1 study
metronidazole 2gm stat day 1+3 - 3 studies
tinidazole 2gm stat - 1 study
clindamycin 2% vag cream with oral clind for 7/7
Mehta, Sex Transm Dis 2012
Absence of evidence of effect is not the same as evidence of absence of effect
Bacterial Response to Treatment
and Biofilms
Biofilms good mechanisms for avoiding
Natural defences, antimicrobials and
Antibiotics
In vivo
Metronidazole temporarily suppressed
biofilm activity during treatment but
activity quickly restarted after treatment
LB gradually fill vacuum formed during treatment
G. vaginalis cleared more slowly then pure anaerobes and levels often
rebound during treatment
BVAB can quickly re-emerge when treatment is stopped
Mayer et al. J Infect Dis 2015
Biofilm persists on vaginal epithelium following lack of response to
treatment
Swidsinski et al. Am J Ob Gyn 2008
Attempts to disrupt the BV biofilm
Many substances tried but either not effective or not
suitable for use in humans so far
In vivo
Metronidazole
Moxifloxacin
Intravaginal lactic acid gel
Intravaginal boric acid
Octenidine (topical antiseptic used in oral biofilms)
In vitro
Synthetic retrocyclin (primate defensin not produced by
humans)
In mice
DNase
Conclusions and where next?
• Bacterial vaginosis is a common condition
• Associated with serious sequelae – increased acquisition
of STIs and HIV, associated with PID and adverse
outcomes in pregnancy
• Cohesive G. vaginalis is probably the main aetiological
agent and is possibly sexually transmitted
• It develops a biofilm to which the other BVAB attach
• The biofilm offers protection to the bacteria and standard
antibiotic treatment is not always successful resulting in
BV recurrence
• We need to find effective and safe BV biofilm-breaking
treatment regimens to improve treatment cure rates
• We need further partner treatment trials to determine if
partner treatment improves cure/reinfection rates